Pollen Viability Stain
Ben Carter, 12/22/15 Adapted from Peterson, Slovin, and Chen, ''Int. J. Plant Biol., ''2010 Anther Harvesting 1. Use forceps to remove a young open flower from the floral bud of a flowering Arabidopsis plant. The objective is to select a flower that is immediately prior to anther dehiscence in which the pollen is mature but not yet shed. Flowers in which the pistil protrudes above the sepals are too old. 2. Stick a piece of double-stick tape to a microscope slide and stick the flower to the tape. Using a stereo microscope and forceps, remove any petals and sepals that obscure your view of the anthers. A flower of the appropriate age will have yellow anthers that are almost the same height as the top of the pistil. If the anthers are not of appropriate maturity, select another flower. Tissue Fixation Gloves should be worn when handling Carnoy's Fixative and associated samples. 3. Using forceps, remove the whole stamens of appropriate maturity from the dissected flower and gently place them in a 1.5mL microcentrifuge tube containing 300µL of Carnoy's Fixative. A minimum of 5 stamens should be collected per sample as some may be damaged during the procedure. 4. Incubate the stamens in the fixative for at least two hours. Samples in fixative can be stored for up to one year at room temperature or 4°C as per Peterson et al. Staining and Imaging 5. Gently pipette away all but about 50µL of the fixative using a P200 or syringe. Using a wide-mouth pipette tip, gently transfer the stamens from the microcentrifuge tube onto a glass microscope slide. Proceed through Step 7 before you begin processing a second sample or the stamens will dry out on the slide. 6. Using a Kimwipe, dab away all excess fixative from the slide surrounding the stamens. Gently dab the stamens to remove any fixative associated with the tissues. Ensure that the stamens are not adhering to the Kimwipe. 7. Add 2-4 drops of Staining Solution over the tissues. If excess fixative remains on the slide, it will become obvious due to the immiscibility of the solutions. Gently hold the microscope slide over the flame of an alcohol burner or Bunsen burner at low heat for about 30 seconds. If using a Bunsen burner, the slide should be moved in and out of the tip of the flame to prevent smoking or bubbling of the stain solution. 8. Place a cover slip over the sample and apply gentle and even pressure to promote the convergence of the tissues into one focal plane. If desired, the slide can be sealed using clear nail polish. Samples can be imaged using a compound light microscope. Aborted pollen grains will appear as blackened masses, while non-aborted pollen grains will appear as purplish spheres. Reference images for various species are available in Peterson et al. Reagents Carnoy's Fixative * 3mL Ethanol * 1.5mL Chloroform * 0.5mL Acetic acid Staining Solution Add ingredients in the order specified. * 5mL 95% Ethanol * 0.5mL 1% Malachite green in 95% ethanol * 25mL Milli-Q water * 12.5mL Glycerol * 2.5mL 1% Acid fuchsin in water * 0.25mL 1% Orange G in water * 2mL Acetic acid * Dilute to 50mL with Milli-Q water. * Store solution in the dark. Category:Arabidopsis Category:Protocols